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Abstract

Transgenic sheep milk containing the protein human alpha 1-Antitrypsin (AAT) was partitioned in Poly(ethylene glycol) (PEG)-Sulphate and PEG-Phosphate biphasic systems. Individual partition coefficients for AAT and some of the milk proteins were determined in these systems. The effects of PEG molecular weight, pH and the inclusion of NaCl on the partitioning of the proteins were also studied. It was found that increasing the concentration of NaCl and decreasing the molecular weight of the PEG resulted in an increase of the partition coefficients of the proteins to the upper (PEG) phase. This partitioning effect was greater for the more hydrophobic proteins and particularly in systems having a pH close to the isoelectric point of the protein. Solubilities of the proteins in increasing concentrations of ammonium sulphate were measured in order to investigate the effects of hydrophobic and electrostatic interactions on the partitioning of these proteins in aqueous two-phase systems. Those proteins that precipitated at low levels of ammonium sulphate showed an increase in partition coefficient at low concentrations of NaCl, or they were precipitated at the interface of the phase at low concentrations of NaCl. Proteins that had low salting out constants in ammonium sulphate solutions were relatively unaffected by NaCl in ATPS. It is probable however that conformational changes and the state of aggregation of proteins are also important and should be invoked in describing the partitioning behavior observed for beta-Lg for example. Comparison of theoretical and experimental values for AAT yield and purity showed clearly that partition coefficients are influenced by the degree of purity and values obtained with purified standards are not necessarily the same as for the same protein present in a complex mixture. Under the most favourable conditions using a 4% w/w loading of transgenic ovine milk, we obtained a 91% yield of AAT in the PEG phase with a purity of 73%.

Partitioning features of bovine trypsin and α-chymotrypsin in polyethyleneglycol-sodium citrate aqueous two-phase systems

“…For both proteins, the presence of salt induced a displacement of the partitioning equilibrium to the top phase. This agrees with a salting out effect in the bottom phase that displaces the equilibrium to the PEG-riched phase [21]. This displacement is less marked for ChTRP since the high ionic strength due to the salt presence also induces a displacement of the aggregation equilibrium to the dimer state [20].…”

Extraction of trypsin from bovine pancreas by applying polyethyleneglycol/sodium citrate aqueous two-phase systems

“…In our case, although PEG3350/NaCit pH 5.20 systems showed to be the most selective ones, they were expected to lead to poor recoveries (24-26%) due to the low K pTRP values. The addition of certain salts, such as NaCl, has been reported [18] to displace the partitioning equilibrium to the top phase, thus enhancing the protein yield in this phase. Fig.…”

Polyethyleneglycol molecular mass and polydispersivity effect on protein partitioning in aqueous two-phase systems

“…This system is frequently used in the isolation of several proteins by the partitioning method [10]. The system pH value 5.6 is given by the concentrated ammonium sulfate solution.…”